Studies will be continued on the conversion and inactivation of neuropeptides with emphasis placed on biosynthetic precursors of opiate peptides (endorphins and enkephalins). Examples of enzymes to be studied include cysteine proteinases (cathepsins B, H and L), angiotensin converting enzyme, a dipeptidyl carboxypeptidase specific for enkephalins, and monocarboxypeptidase (cathepsin A). Cysteine proteinases will be used to pinpoint their sites of cleavage using beta-lipotropin (LPH 1-91) as substrate, or beta-endorphin (LPH 61-91) and the products separated by reverse phase high pressure liquid chromatography (HPLC). Dipeptidyl carboxypeptidases will be examined for their role in the conversion of enkephalins having C-terminal extensions as found in dynorphin, alpha-neo-endorphin, Met-enkephalin-Arg6, and Met-enkephalin-Arg6-Phe7 (or the same hexa- and heptapeptide with Leu instead of Met in position in 5). Cathepsin A will be examined to see if it can account for the production of hexapeptide forms found in striatum by conversion of hepatapeptide. Angiotensin converting enzyme will be purified by immunoaffinity chromatography and separated from the specific enkephalinase(s) that release also Phe-Met from pentapeptide enkephalins. An attempt will be made to prepare homogenous forms of enkephalinase free of exopeptidase or other enzyme contamination in order to investigate their role in conversion processes. Cathepsins B, L and H will separate by CM-cellex chromatography and then purified further by affinity chromatography on thiol sepharoses.